Not applicable.
The invention provides isolated nucleic acid and amino acid sequences of Kv6.2, antibodies to Kv6.2, methods of detecting Kv6.2, methods of screening for voltage-gated potassium channel activators and inhibitors using biologically active Kv6.2, and kits for screening for activators and inhibitors of voltage gated potassium channels comprising Kv6.2.
Potassium channels are involved in a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Potassium channels are thus found in a wide variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are made by alpha subunits that fall into 8 families, based on predicted structural and functional similarities (Wei et al., Neuropharmacology 35(7):805-829 (1997)). Three of these families (Kv, Eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families, CNG and SK/IK, also contain this motif but are gated by cyclic nucleotides and calcium, respectively. The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25):14066-71 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al., J. Biol. Chem. 273:3509-16 (1998)). Another family, the inward rectifier potassium channels (Kir), belong to a structural family containing 2 transmembrane domains (see, e.g., Lagrutta et al., Jpn. Heart. J. 37:651-660 1996)), and an eighth functionally diverse family (TP, or xe2x80x9ctwo-porexe2x80x9d) contains 2 tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels made from Kv, KQT and Slo or BK subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These beta subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels. formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al., J. Physiol. 493:625-633 (1996); Shi et al., Neuron 16(4):843-852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al., Nature 384:80-83 (1996)).
The Kv superfamily of voltage-gated potassium channels includes both heteromeric and homomeric channels that are typically composed of four subunits (see, e.g., Salinas et al., J. Biol. Chem. 272:8774-8780 (1997); Salinas et al., J. Biol. Chem. 272:24371-24379 (1997); Post et al., FEBS Letts. 399:177-182 (1996)). Voltage-gated potassium channels have been found in a wide variety of tissues and cell types and are involved in processes such as neuronal integration, cardiac pacemaking, muscle contraction, hormone section, cell volume regulation, lymphocyte differentiation, and cell proliferation (see, e.g., Salinas et al., J. Biol. Chem. 39:24371-24379 (1997)). Some alpha subunits of the Kv superfamily, of which the channels are composed, have been cloned and expressed, e.g., Kv5.1, Kv6.1 (Drewe et al., J. Neurosci. 12:538-548 (1992); Post et al., FEBS Letts. 399:177-182 (1996)); Kv8.1 (Hugnot et al., EMBO J. 15:3322-3331 (1996)); and Kv9.1 and 9.2 (Salinas et al., J. Biol. Chem. 39:24371-24379 (1997)). Expression patterns of some of these genes has also been examined (see, e.g., Verma-Kurvari et al., Mol. Brain. Res. 46:54-62 (1997); Maletic-Savatic et al., J. Neurosci. 15:3840-3851 (1995); Du et al., Neurosci. 84:37-48 (1998)).
The present invention thus provides for the first time Kv6.2, a polypeptide monomer that is an alpha subunit of an heteromeric voltage-gated potassium channel. Kv6.2 has not been previously cloned or identified, and the present invention provides the nucleotide and amino acid sequences for mouse and human Kv6.2.
In one aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having a monomer subunit association region that has greater than 70% amino acid sequence identity to a Kv6.2 subunit association region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In one aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having an S4-S6 region that has greater than 85% amino acid sequence identity to a Kv6.2 S4-S6 region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In one embodiment, the nucleic acid encodes mouse or human Kv6.2. In another embodiment, the nucleic acid encodes SEQ ID NO:1 or SEQ ID NO: 17. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:18.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of:
ATGCCCATGTCTTCCAGAGACAGG (SEQ ID NO:3), GATGTCTAGAGGGAGTTACATGTAGCG (SEQ ID NO:4) and GGCACTACGCATCCTCTACGTAATGCGC (SEQ ID NO:5), GATGATGGCCCACCAATAGGATGCGG (SEQ ID NO:6) and ATGCCCATGCCTTCCAGAGACGG (SEQ ID NO:7), TTACATGTGCATGATAGGCAAGGCTG (SEQ ID NO:8) and GTCCAGGCCCAAGACAAGTGTCAG (SEQ ID NO:9), GGGAGAAGGTGTGGAAGATAGACG (SEQ ID NO:10).
In one embodiment, the nucleic acid encodes a polypeptide monomer having a molecular weight of between about 53 kDa to about 65 kDa. In one embodiment, the nucleic acid selectively hybridizes under moderately stringent hybridization conditions to a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:18.
In another aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer, wherein the nucleic acid specifically hybridizes under highly stringent conditions to SEQ ID NO:2 or SEQ ID NO:18.
In another aspect, the present invention provides an isolated polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the potassium channel: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having a monomer subunit association region that has greater than 70% amino acid sequence identity to amino acids a Kv6.2 subunit association region; and (iv) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In another aspect, the present invention provides an isolated polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the potassium channel: (i) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (ii) having an S4-S6 region that has greater than 85% amino acid sequence identity to a Kv6.2 S4-S6 region; and (iv) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17.
In one embodiment, the polypeptide monomer has an amino acid sequence of mouse or human Kv6.2. In another embodiment, the polypeptide monomer has an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:17.
In another aspect, the present invention provides an antibody that selectively binds to the polypeptide monomer described above.
In another aspect, the present invention provides an expression vector comprising the nucleic acid encoding the polypeptide monomer described above.
In another aspect, the present invention provides a host cell transfected with the expression vector described above.
In another aspect, the present invention provides a method for identifying a compound that increases or decreases ion flux through an voltage-gated potassium channel, the method comprising the steps of: (i) contacting the compound with a eukaryotic host cell or cell membrane in which has been expressed a polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the polypeptide monomer: (a) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (b) having a monomer subunit association region that has greater than 70% amino acid sequence identity to a Kv6.2 subunit association region; and (c) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17; and (ii) determining the functional effect of the compound upon the cell or cell membrane expressing the potassium channel.
In another aspect, the present invention provides a method for identifying a compound that increases or decreases ion flux through an voltage-gated potassium channel, the method comprising the steps of: (i) contacting the compound with a eukaryotic host cell or cell membrane in which has been expressed a polypeptide monomer comprising an alpha subunit of a heteromeric potassium channel, the polypeptide monomer: (a) having the ability to form, with at least one additional Kv alpha subunit, a heteromeric potassium channel having the characteristic of voltage gating; (b) having an S4-S6 region that has greater than 85% amino acid sequence identity to a Kv6.2 S4-S6 region as measured using a sequence comparison algorithm; and (c) specifically binding to polyclonal antibodies generated against SEQ ID NO:1 or SEQ ID NO:17; and (ii) determining the functional effect of the compound upon the cell or cell membrane expressing the potassium channel.
In one embodiment, the increased or decreased flux of ions is determined by measuring changes in current or voltage. In another embodiment, the polypeptide monomer polypeptide is recombinant.
In another embodiment, the present invention provides a method of detecting the presence of Kv6.2 in mammalian tissue, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a Kv6.2-specific reagent that selectively associates with Kv6.2; and, (iii) detecting the level of Kv6.2-specific reagent that selectively associates with the sample.
In one embodiment, the Kv6.2-specific reagent is selected from the group consisting of: Kv6.2 specific antibodies, Kv6.2 specific oligonucleotide primers, and Kv6.2 nucleic acid probes. In another embodiment, the sample is from a human.
In another aspect, the present invention provides, in a computer system, a method of screening for mutations of Kv6.2 genes, the method comprising the steps of: (i) entering into the computer a first nucleic acid sequence encoding an voltage-gated potassium channel protein having a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:18, and conservatively modified versions thereof; (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity to the first nucleic acid sequence; and (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In one embodiment, the second nucleic acid sequence is associated with a disease state.
In another aspect, the present invention provides, in a computer system, a method for identifying a three-dimensional structure of Kv6.2 polypeptides, the method comprising the steps of: (i) entering into the computer system an amino acid sequence of at least 25 amino acids of a potassium channel monomer or at least 75 nucleotides of a gene encoding the polypeptide, the polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:17, and conservatively modified versions thereof; and (ii) generating a three-dimensional structure of the polypeptide encoded by the amino acid sequence.
In one embodiment, the amino acid sequence is a primary structure and wherein said generating step includes the steps of: (i) forming a secondary structure from said primary structure using energy terms determined by the primary structure; and (ii) forming a tertiary structure from said secondary structure using energy terms determined by said secondary structure. In another embodiment, the generating step includes the step of forming a quaternary structure from said tertiary structure using anisotropic terms determined by the tertiary structure. In another embodiment, the methods further comprises the step of identifying regions of the three-dimensional structure of the protein that bind to ligands and using the regions to identify ligands that bind to the protein.